With advances in gene therapy technologies, the use of therapeutic viral vectors represents an increasingly effective paradigm for treating human diseases. Among the viral vectors available for gene therapy applications are lentiviral vectors. Such vectors include reconstructed viral vector systems derived from human immunodeficiency virus-1 (HIV-1) and are capable of introducing a gene of interest into animal and human primary cells or cell lines. The genomes of lentiviral vectors include a coding strand of RNA, which is reverse-transcribed into DNA upon entering the cytoplasm of a host cell by a viral reverse transcriptase so as to form a DNA pre-integration complex. This complex is transported into the nucleus of the host cell, where a portion of the viral DNA is subsequently integrated into the host cell genome. The integrated DNA can then be transcribed into RNA, such as protein-coding mRNA, which can ultimately be exported to the cytoplasm for subsequent expression of a protein of interest.
Lentiviral vector-mediated gene expression can be used to achieve continuous and stable protein production, because the gene of interest has been integrated into a host cell's genome and is thus replicated upon division of the cell. Lentiviral vectors can effectively infect non-dividing cells as well as those actively progressing through the cell cycle. In contrast, other viral vectors, such as adenoviral vectors, adeno-associated viral vectors, and classical retroviral vectors, are only capable of infecting dividing cells. Tissues and cells in which lentiviral vector-mediated chronic expression of a gene of interest can occur include the brain, liver, muscle cells, retina, hematopoietic stem cells, marrow mesenchymal stem cells, and macrophages, among others.
The production of lentiviral vectors has been hindered by several challenges, one of which is low stability of the vectors. The manufacturing operation of lentiviral vectors includes several steps: production, purification, storage, and application of gene transfer (Carmo et al., J. Gene Med. 11:670-678, 2009). Lentiviral vectors are susceptible to inactivation during these processes, which can contribute to diminished final quality and efficacy of the vector preparation. In previous studies, it has been shown that one mechanism by which viral vectors are inactivated is by the loss of viral capacity to perform reverse transcription (Carmo et al., Hum. Gene Ther. 20:1168-76, 2009; and Carmo et al., J. Gene Med. 10:383-391, 2008). Moreover, there remains a need for methods to stabilize lentiviral vector preparations so as to prevent irreversible aggregation that can be accompanied by loss of infectivity. Additionally, during the purification of lentiviral vectors, stabilizing components are removed from the lentiviral preparation, which can cause the vector to become increasingly unstable. Therefore, there is also a need for lentiviral formulations that preserve vector stability throughout the purification process.
During purification and storage, vectors are often stored at 4° C. (Rodrigues et al., J. Biotechnol. 127:520-541, 2007). It has been reported that lentiviral vectors have an additional need for stabilizing components, such as human serum albumin (HSA) (Carmo et al., J. Gene Med. 11:670-678, 2009). This is in sharp contrast to gamma-retroviruses, where simply adding exogenous proteins brings back the stability comparable to cell culture supernatant. Lipoproteins are complex structures composed of several lipids, including cholesterol, phospholipids, and proteins (Olson, J. Nutr. 128:S439-S443, 1998). They act as lipid transporters in blood along with HSA. It is possible that a lipoprotein-HSA structure forms a protective arrangement around the membrane of lentiviral vectors (Carmo et al., J. Gene Med. 11:670-678, 2009). Because albumin is also known to associate tightly with cell surfaces (Dziarski et al., J. Biol. Chem. 269:20431-20436, 1994), these lipoprotein/HSA complexes can associate with the membrane of the vector, which is similar to a cell membrane. This association may provide protection to their structure and prevent conformational changes more efficiently than HSA alone.
In order to ensure stability during storage, stocks of infective viral vectors have commonly been stored at low temperatures (e.g., at −80° C.) due to their complexity. It has been suggested that lipid-enveloped viruses survive well at temperatures below −60° C., and that storage at −20° C. or 4° C. should only be used if “retention of virus infectivity is not essential” (Gould et al., Mol. Biotechnol. 13:57-66, 1999). Other investigations have concluded that certain viral vectors should be stored at −70° C. or lower in order to retain infectivity (Harper, Virology Ed. BIOS Scientific Publishers Limited, Oxford, UK, 1993). Typically, lentiviral vector preparations contain proteins encoded by the viral genome, including envelope proteins embedded in a lipid bilayer membrane. At low temperatures, the protein can be susceptible to denaturation and the lipid bilayer may be prone to loss of structural integrity. Therefore, a need exists for lentiviral formulations capable of increasing the stability of a viral vector preparation at a low temperature, for extended periods of time.
Lentiviral vectors are often maintained at these low temperatures for long-term storage, as iterative freezing and thawing of viral vectors can lead to a loss of transducing capacity. As such, there remains a need for lentiviral preparations that retain infectivity after undergoing multiple freeze/thaw cycles.
In addition to stability during purification and storage, a lentiviral vector useful for ex vivo applications, such as chimeric antigen receptor T (CART) cell therapy, desirably will retain stability at physiologically relevant temperatures, such as 37° C., the temperature at which lentiviral vectors may be incubated with host cells in order to promote transduction. Therefore, there also exists a need for lentiviral preparations that maintain structural integrity of the viral vector during gene transfer events ex vivo.
In addition to the above-noted biological considerations, lentiviral vector preparations that preserve viral vector stability can additionally be useful from a commercial perspective. When a recombinant lentiviral vector is stored at low temperatures for excessive periods of time or when the recombinant vector undergoes multiple freeze/thaw cycles during experimental use or manufacturing operations, the biological activity decreases significantly. This leads to a diminished recovery of infectious particles, further raising the cost of goods (COGs). In addition, a higher susceptibility of vector particles to lose activity can lead to inaccurate results in preclinical or clinical studies. For a clinical setting, use of an ultra-low temperature (e.g., −60° C. or below) storage device is an additional cost burden and poses a logistical challenge in hospitals and other point-of-care facilities. Generally, these facilities are expected to have an ultra-low freezing apparatus to deliver the treatment. Therefore, there remains a need for recombinant lentiviral vector preparations that preserve vector stability so as to promote efficient manufacturing operations and viable low-temperature storage methods.